Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFβ1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFβ1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFβ1 mRNA expression and protein translation. Transfection of TGFβAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFβ1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNβ, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFβAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2’-5’-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFβAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFβ expression. These findings also suggest TGFβ as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-023-03760-8.


Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) causes global economic loss of swine industry.PRRSV is an enveloped RNA virus under family Arteriviridae, order Nidovirales.Its genome is approximately 15 kb in size, consisting of 11 open reading frames (ORFs).The virus is classified as PRRSV-1 (formerly European genotype) and PRRSV-2 (formerly North American genotype).Both PRRSV species share up to 60% nucleotide sequence homology and comprise classical PRRSV (cPRRSV) strains and highly pathogenic PRRSV (HP-PRRSV) strains [1].
In this study, we aim to investigate the effects of PRRSV-induced TGFβ overexpression on immunerelated gene responses in PRRSV-inoculated MDMs.We employed phosphorothioate-modified antisense (AS) oligodeoxynucleotides (ODNs) specific for porcine TGFβ1 mRNA to knock down its expression.Our findings report that TGFβ plays roles in down-regulating gene expressions of co-stimulatory molecules, type I IFN, IRGs, and pro-inflammatory cytokines in PRRSV-inoculated MDMs.Our findings suggest potential strategies to improve innate and adaptive CMI responses to future PRRSV vaccines and vaccine adjuvants.

Animals
Eight 24-week-old PRRSV-seronegative crossbred pigs (Large White/Landrace x Duroc) were the sources of PBMCs.They were housed at the swine research farm, faculty of animal science and technology, Maejo University.

Phosphorothioate-modified ODNs
All phosphorothioate-modified ODNs were synthesized by Integrated DNA Technologies (IDT, Singapore).Their sequences are detailed in Table 1.
Total RNA was isolated using a NucleoSpin® RNA kit (Macherey-Nagel, Germany).The quality and quantity of RNA were evaluated by a Nanodrop 2000/2000c spectrophotometer (NanoDrop Technologies, Montchamin, DE).All RNA samples had A260/230 and A260/280 between 1.8 and 2.2 and 2.0-2.2,respectively.The integrity of RNA was determined by denaturing agarose gel electrophoresis followed by ethidium bromide staining.Reverse transcription was carried out, using RevertAid™ First Strand cDNA synthesis kit (Thermoscientific, Lithuania).The reaction used 1,000 ng of pooled total RNA as template, and a mixture of oligo-dT and random hexamers as primers.cDNA was stored at -20 °C until real-time polymerase chain reaction (PCR).

Transfection of MDMs with AS-ODNs
Transfection was carried out following the guideline of Lipofectamine ™ RNAiMax (Invitrogen, Carlsbad, CA) with recommended small interfering RNA (siRNA, BLOCK-iT™ Alexa Fluor® Red Fluorescent control, Invitrogen).In brief, different mixtures of Lipofectamine ™ RNAiMax in Opti-MEM® I (v/v) and 2 µM siRNA suspended in Opti-MEM® I were added to the wells containing MDMs.Cell uptake of fluorescent-labeled siRNA was observed under the immunofluorescent microscope (Nikon Eclipse Ti, Japan).Frequencies of immunofluorescent-positive cells were identified using automatic measurement for cell counting (NIS-elements software ver.3.22, Nikon, Japan).Cell viability was determined by trypan blue staining in parallel.Optimal concentration of transfection reagent and optimal transfection period were determined.

Evaluation of TGFβAS1-4 efficiency on TGFβ1 mRNA knockdown
TGFβAS mixtures containing 2 µM of either TGFβAS1, TGFβAS2, TGFβAS3, or TGFβAS4 in transfection media (1.5% v/v of Lipofectamine™ RNAiMAX in Opti-MEM® I) were incubated at RT for 30 min.Then, 20 µl of the mixtures were added to wells containing MDMs in 100 µl of Opti-MEM® I.The cultures were mixed gently by rocking the plates back and forth for 5 min and incubated further for 4 h (37 o C, humidified 5% CO 2 ).The media were then removed and replaced with 200 µl of RPMI ++ and 50 µl of inducers.Plates were incubated for 12 h (37 o C, humidified 5% CO 2 ), then the cells were harvested, washed with PBS, and evaluated for TGFβ1 mRNA expression by realtime PCR.Untransfected MDMs stimulated or not with inducers served as positive and negative controls, respectively.MDMs treated with scramble (Scr) ODNs or transfection media prior to stimulation with inducers served as Scr and transfection media controls, respectively.For the determination of mRNA expression levels of TGFβ1 and other immune-related genes, 200 ng of total RNA was used as the template for cDNA synthesis.The threshold cycles (C T ) of all genes were used for the calculation of gene expression by 2^(-ΔΔC T ) method.The expressions of TGFβ1 and other immune-related genes were normalized to the geometric average of RPL32 (ribosomal protein L32) and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta) and calibrated to that in the negative control.High expression stability of RPL32 and YWHAZ in porcine MDMs stimulated or not with inducers has been reported [31].The expression levels of all immunerelated genes were transformed into log 2 scale.

Evaluation of TGFβ1 knockdown effects on immunerelated gene expressions in MDMs inoculated with cPRRSV-2 and HP-PRRSV-2
TGFβAS1 (2 µM) in transfection media were transfected to MDMs as described above.Subsequently, transfection media were removed and replaced with 100 µl RPMI ++ and 100 µl of either cPRRSV-2 or HP-PRRSV-2 (equivalent to multiplicity of infection (m.o.i) of 1).The cultures were incubated for 48 h (37 °C, humidified 5% CO 2 ), then received 50 µl of inducers.The cultures were incubated further for 12 h (37 °C, humidified 5% CO 2 ) prior to RNA isolation.Cell culture supernatants were collected for the determination of TGFβ1 protein levels by ELISA.Expressions of immune-related genes were determined by real-time PCR.Controls included MDMs receiving mock Ag plus inducers (mock control); MDMs receiving PRRSV-2 and inducers (PRRSV-2-inoculated control); and MDMs treated with transfection media alone (without TGFβAS1), inoculated with PRRSV-2, and stimulated with inducers (PRRSV-2-inoculated/transfection media control).Untreated MDMs receiving culture media in the presence or absence of inducers served as positive and negative controls, respectively.Cell viability was determined at the end of the transfection period, PRRSV-2 inoculation, and inducer stimulation using trypan blue.
PRRSV-2 RNA was isolated and contaminating DNA was eliminated, using Nucleospin® RNA virus kit and rDNase (both from Macherey-Nagel), respectively.The quality and quantity of RNA were evaluated by Nanodrop 2000/2000c spectrophotometer.Reverse transcription (using RevertAid™ First Strand cDNA synthesis kit) and real-time PCR were conducted as described previously [32].In brief, a total reaction volume of 20 µl, consisting of 2 µl cDNA, 10 µl SYBR® Green PCR master mix (Toyobo), and 400 nM each of primer ORF7 149 F and ORF7 346R was set up in duplicate.The PCR condition was 95 °C (15 min); and 35 cycles of 95 °C (15 s), 53 °C (30 s), and 72 °C (30 s).The C T was collected and compared with the standard curve of C T generated from 10 1 to 10 8 copy numbers of recombinant PRRSV-2 ORF7 plasmids.Melting curve analysis and agarose gel electrophoresis were performed to verify a single product.A nuclease-free water was included as no template control in every run.

Statistical analysis
Statistical analyses were performed using the SPSS software version 17 (IBM, Armonk, NY).Mean differences of immune-related gene expression levels and TGFβ1 protein levels among groups were tested by one-way ANOVA, followed by Tukey HSD test.Mean differences of percentages of fluoresced cells and PRRSV-2 ORF7 RNA copy numbers among groups were tested by oneway repeated measures ANOVA, followed by Tukey HSD test.P < 0.05 was set as a statistically significant level.

TGFβAS1 efficiently knockdown TGFβ1 mRNA expression
Using fluorescent-labeled siRNA control, 1.5% (v/v) transfection media and a transfection period of 4 h yielded the highest transfection efficiency with approximately 60% fluorescent-positive MDMs (Fig. 1B and C).These conditions were therefore used for subsequent transfection experiments.
Phosphorotioate-modified AS ODNs were designed to target various regions of porcine TGFβ1 mRNA (Table 1).Activity-decreasing motifs, i.e., GGGG, ACTG, AAA, TAA, and CCGG were absent from all AS ODNs to ensure the antisense activity.Using optimized transfection conditions, transfection with TGFβAS1, which targeted the AUG region of porcine TGFβ1 mRNA, efficiently knockdown TGFβ1 mRNA expression compared to positive control (Fig. 1D).No knockdown effect on TGFβ1 mRNA expression was observed in MDMs transfected with TGFβAS2-4, TGFβS1-4, and Scr1-4 (Fig. 1D).The knockdown effect of TGFβAS1 was dose-dependent as the effect was observed more strongly at 2 µM than at 1 and 0.5 µM, respectively (Fig. 1E).Significant reduction of TGFβ1 protein level was also detected in TGFβAS1transfected MDMs (Fig. 1F).

TGFβAS1 was specific to TGFβ1 mRNA and did not knockdown mRNA expression of any other immunerelated genes
The specificity of TGFβAS1 was evaluated by analyzing with BLAST search for its potential target to porcine immune-related genes other than TGFβ1.The TGFβAS1 had no aligned target in any of the immune-related genes presented in this study or essential genes involved in swine immune system (data not shown).The TGFβAS1 also had no aligned target in any ORFs of cPRRSV-2 and HP-PRRSV-2 used in this study (data not shown).

TGFβAS1 significantly knockdown TGFβ1 mRNA expression which was up-regulated by cPRRSV-2 and HP-PRRSV-2 and contributed to improving gene expressions of co-stimulatory molecules, type I IFN, IRGs, and proinflammatory cytokine which were down-regulated by the viruses
Compared to positive control, MDMs inoculated with cPRRSV-2 and HP-PRRSV-2 demonstrated significantly increased TGFβ1, TLR7, and TLR8 mRNA expressions, and significantly reduced CD80, CD86, IFNα, IFNβ, IFNγ, IRF3, IRF7, Mx1, OAS1, OPN, STING, and TNFα Fig. 2 Heat map illustrating expression levels of immune-related genes in MDMs transfected with either TGFβAS1 (I) or Scr1 (II), or otherwise treated with transfection media (III) alone prior to stimulation with a mixture of poly I:C and LPS.Untransfected MDMs stimulated with a mixture of poly I:C and LPS served as positive control (IV).Data were normalized to the geometric average of RPL32 and YWHAZ relative to untransfected/unstimulated MDMs.Data are presented in log 2 scale of "fold" according to 2^(-ΔΔC T ) method mRNA expressions (Fig. 3 and Supplementary Table 3).MDMs inoculated with HP-PRRSV-2 also demonstrated significantly increased IL-1β and TLR3 mRNA expressions.MDMs inoculated with HP-PRRSV-2 showed significantly higher mRNA expressions of TGFβ1, TLR3, and TLR8, and significantly lower mRNA expressions of IFNα, IFNβ, IFNγ, IRF3, Mx1, OPN, and TNFα than MDMs inoculated with cPRRSV-2.Mock Ag had no effect on the modulation of mRNA expressions of these immune-related genes.

IFNα significantly contributed to the reduced amount of cPRRSV-2 and HP-PRRSV-2 RNA yields
Since TGFβ1 knockdown resulted in significantly increased expressions of co-stimulatory molecules, type I IFN, IRGs, TLR3, and TNFα, and significantly reduced amounts of PRRSV-2 ORF7 RNA copy numbers, it is of interest to investigate further the direct effect of those immune-related molecules on PRRSV-2 RNA yields.For such investigation, commercially available rIFNα was chosen as a candidate.

Discussion
This study investigated the effects of PRRSV-induced TGFβ overexpression on mRNA expressions of co-stimulatory molecules, type I IFN, IRGs, pattern recognition receptors, and pro-inflammatory cytokines in PRRSVinoculated MDMs.Up-regulation of TGFβ expression has been reported in PRRSV-infected cells, e.g.MDMs and PBMCs, and in lymphoid organs and lungs of PRRSV-infected pigs [18][19][20].To date, the role of PRRSV-up-regulated TGFβ expression on immune protection against PRRSV has not yet been studied.
Among all four sequences of phosphorotioate-modified TGFβAS ODNs (Table 1), only those that target AUG region of TGFβ1 mRNA significantly reduced TGFβ1 mRNA expression and protein translation (Fig. 1D-F).In pigs, AUG region has been reported to be a potential target for gene knockdown of at least two cytokines, i.e.IL-10 and IFNγ [33].The phosphorothioate-modified AS ODNs, theoretically, control target mRNA expression by binding specifically to target mRNA region, and forming mRNA/AS ODN duplexes which then trigger RnaseH to cleave the hybridized target mRNA [34].This results in reduced amounts or absence of intact mRNA template for translation, and thereby reduced target protein level.
Significantly increased mRNA expressions of TGFβ1, IL-1β, TLR3, TLR7, and TLR8 were detected in MDMs inoculated with cPRRSV-2 and HP-PRRSV-2 (Fig. 3 and Supplementary Table 3).The levels of mRNA expressions of TGFβ1, TLR3, and TLR8 were higher in HP-PRRSV-2-inoculated MDMs than in cPRRSV-2-inoculated MDMs (Fig. 3 and Supplementary Table 3).No change in TLR4 and TLR9 mRNA expression was detected.Similar findings have been reported in pulmonary alveolar macrophages (PAMs) of PRRSV-infected pigs that PAMs from HP-PRRSV-2-infected pigs expressed higher levels of TLR3, TLR7, TLR8, and IL-1β mRNA than PAMs from cPRRSV-2-infected pigs [35].The up-regulation of TLR3, TLR7, and TLR8 mRNA expression reportedly followed the initial down-regulation of mRNA expression of these genes in PRRSV-infected PAMs and immature DCs [3].In pigs, up-regulation of TLR3, TLR7, and TLR8 mRNA expression has been reported in lymphoid tissues following PRRSV infection, which was proposedly associated with the increased susceptibility of pigs to secondary infections and the increased severity of the diseases [36].
To elucidate further whether PRRSV-up-regulated TGFβ1 expression supports PRRSV replication, rTGFβ1 was used.Treatment of MDMs with rTGFβ1 prior to either cPRRSV-2 or HP-PRRSV-2 inoculation significantly increased PRRSV-2 ORF7 RNA copy numbers (Fig. 7).Treatment of MDMs with rTGFβ1 prior to rIFNα treatment and cPRRSV-2 or HP-PRRSV-2 inoculation reduced the antiviral activity of rIFNα (Fig. 7).These findings clearly indicate the positive role of TGFβ1 on PRRSV replication.These findings also suggest a strategy of PRRSV to enhance virus replication and reduce innate immune defense against the virus through an up-regulation of TGFβ1 expression.

Conclusion
Both cPRRSV-2 and HP-PRRSV-2 significantly induced TGFβ1 mRNA expression in MDMs.TGFβ1 protein translation in MDMs was significantly induced by HP-PRRSV-2.Knockdown of TGFβ1 expression by TGFβAS1 significantly improved mRNA expression levels of CD80, CD86, IFNβ, IRGs (i.e.IRF3, IRF7, Mx1, OPN, STING), TLR3, and TNFα in MDMs inoculated with the virus.Knockdown of TGFβ1 expression by TGFβAS1 significantly contributed to the reduced yields of PRRSV-2 RNA copy numbers.On the contrary, recombinant TGFβ1 sustained the yields of PRRSV-2 RNA copy numbers.These findings demonstrate a potential innate immune suppressive strategy of PRRSV and the immunomodulatory role of PRRSV-induced TGFβ on downmodulating innate immune defense against the virus.These findings also suggest a potential target that a development of future PRRSV vaccines and vaccine adjuvants should take into consideration.

Fig. 1
Fig. 1 Optimization and validation of knockdown of porcine TGFβ1 mRNA expression and protein translation by AS ODNs.A) MDMs under bright-field microscopy.B) MDMs uptake of fluorescent-labeled siRNA under immunofluorescent microscopy.C) MDMs uptake of fluorescent-labeled siRNA complexed with different concentrations of transfection reagent and transfection period.D) Effect of TGFβ1 antisense (AS1-4), sense (S1-4) and scramble (Scr1-4) phosphorothioate-modified ODNs on the expression of TGFβ1 mRNA in MDMs stimulated with a mixture of poly I:C and LPS.Band intensities (Additional file 1) indicate the quality of TGFβ1 knockdown.E) Optimization of TGFβAS1 concentration for TGFβ1 mRNA knockdown on MDMs transfected with TGFβAS1 (0.5, 1, or 2 µM) and stimulated with a mixture of poly I:C and LPS.Band intensities (Additional file 2) indicate the quality of TGFβ1 knockdown.F) TGFβ1 protein levels in MDMs transfected with TGFβAS1 (2 µM) and stimulated with a mixture of poly I:C and LPS.In all figures, error bars indicate the standard deviation (SD).Mean differences of TGFβ1 gene expression or protein translation among groups were tested by one-way ANOVA, followed by Tukey HSD test.Mean differences of percentages of fluoresced cells among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD test.Different letters indicate significant differences.P < 0.05 was set as a statistically significant level

Fig. 4
Fig. 4 Effect of TGFβAS1 on TGFβ1 protein translation in PRRSV-2-inoculated MDMs.MDMs were transfected with TGFβAS1, then inoculated with either cPRRSV-2 or HP-PRRSV-2, and stimulated with a mixture of poly I:C and LPS.MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control.MDMs treated with transfection media (Tr.media) and inoculated with cPRRSV-2 or HP-PRRSV-2, then stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated/Tr.media control.MDMs inoculated with mock Ag and stimulated with a mixture of poly I:C and LPS served as mock control.Untreated MDMs receiving culture media in the presence or absence of a mixture of poly I:C and LPS served as positive and negative controls, respectively.Cell culture supernatants were collected for ELISA.Error bars indicate the SD.Mean differences of TGFβ1 protein translation among groups were tested by one-way ANOVA, followed by Tukey HSD test.Different letters indicate significant differences.P < 0.05 was set as a statistically significant level

Fig. 6 Fig. 5
Fig.6 Effects of rIFNα on PRRSV-2 ORF7 RNA copy numbers in PRRSV-2-inoculated MDMs.MDMs were treated with rIFNα (10, 1 and 0.1 ng/ml final), then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h).MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control.MDMs receiving mock Ag plus a mixture of poly I:C and LPS served as uninoculated control.Cell culture supernatants were collected for real-time PCR.The C T values were obtained and PRRSV-2 ORF7 RNA copy numbers were calculated based on the C T standard curve generated from 10 1 -10 8 copies of recombinant PRRSV-2 ORF7 plasmids.Data were presented in log 10 scale of copy number/mL.Error bars indicate the SD.Mean differences of PRRSV-2 ORF7 RNA copy numbers among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD.Different superscript letters indicate significant differences.P < 0.05 was set as a statistically significant level

Fig. 7
Fig.7Effects of rTGFβ1 and rIFNα on PRRSV-2 ORF7 RNA copy numbers in PRRSV-2-inoculated MDMs.MDMs were treated with rTGFβ1 (10 ng/ml final), followed by rIFNα (10 ng/ml final), then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h).MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control.MDMs treated with rTGFβ1, then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h) served as rTGFβ1-treated/ PRRSV-2-inoculated control.MDMs treated with rIFNα, then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h) served as rIFNα -treated/PRRSV-2-inoculated control.MDMs receiving mock Ag plus a mixture of poly I:C and LPS served as uninoculated control.Cell culture supernatants were collected for real-time PCR.The C T values were obtained and PRRSV-2 ORF7 RNA copy numbers were calculated based on the C T standard curve generated from 10 1 -10 8 copies of recombinant PRRSV-2 ORF7 plasmids.Data were presented in log 10 scale of copy number/mL.Error bars indicate the SD.Mean differences of PRRSV-2 ORF7 RNA copy numbers among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD.Different superscript letters indicate significant differences.P < 0.05 was set as a statistically significant level

Table 1
Sequences of antisense (AS), sense (S), and scramble (Scr) phosphorothioate-modified ODNs used in this